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Correct regulation of intercellular communication is a fundamental requirement for cell differentiation. In Arabidopsis thaliana, the female germline differentiates from a single somatic ovule cell that becomes encased in ß-1,3-glucan, a water insoluble polysaccharide implicated in limiting pathogen invasion, regulating intercellular trafficking in roots, and promoting pollen development. Whether ß-1,3-glucan facilitates germline isolation and development has remained contentious, since limited evidence is available to support a functional role. Here, transcriptional profiling of adjoining germline and somatic cells revealed differences in gene expression related to ß-1,3-glucan metabolism and signalling through intercellular channels (plasmodesmata). Dominant expression of a ß-1,3-glucanase in the female germline transiently perturbed ß-1,3-glucan deposits, allowed intercellular movement of tracer molecules, and led to changes in germline gene expression and histone marks, eventually leading to termination of germline development. Our findings indicate that germline ß-1,3-glucan fulfils a functional role in the ovule by insulating the primary germline cell, and thereby determines the success of downstream female gametogenesis.

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Apomixis, or asexual seed formation, is prevalent in Citrinae via a mechanism termed nucellar or adventitious embryony. Here, multiple embryos of a maternal genotype form directly from nucellar cells in the ovule and can outcompete the developing zygotic embryo as they utilize the sexually derived endosperm for growth. Whilst nucellar embryony enables the propagation of clonal plants of maternal genetic constitution, it is also a barrier to effective breeding through hybridization. To address the genetics and evolution of apomixis in Citrinae, a chromosome-level genome of the Hongkong kumquat (Fortunella hindsii) was assembled following a genome-wide variation map including structural variants (SVs) based on 234 Citrinae accessions. This map revealed that hybrid citrus cultivars shelter genome-wide deleterious mutations and SVs into heterozygous states free from recessive selection, which may explain the capability of nucellar embryony in most cultivars during Citrinae diversification. Analyses revealed that parallel evolution may explain the repeated origin of apomixis in different genera of Citrinae. Within Fortunella, we found that apomixis of some varieties originated via introgression. In apomictic Fortunella, the locus associated with apomixis contains the FhRWP gene, encoding an RWP-RK domain-containing protein previously shown to be required for nucellar embryogenesis in Citrus. We found the heterozygous SV in the FhRWP and CitRWP promoters from apomictic Citrus and Fortunella, due to either two or three miniature inverted transposon element (MITE) insertions. A transcription factor, FhARID, encoding an AT-rich interaction domain-containing protein binds to the MITEs in the promoter of apomictic varieties, which facilitates induction of nucellar embryogenesis. This study provides evolutionary genomic and molecular insights into apomixis in Citrinae and has potential ramifications for citrus breeding.

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In most diploids the centromere-specific histone H3 (CENH3), the assembly site of active centromeres, is encoded by a single copy gene. Persistance of two CENH3 paralogs in diploids species raises the possibility of subfunctionalization. Here we analysed both CENH3 genes of the  diploid dryland crop cowpea. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of Vigna unguiculata. Both functional CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development. Hence, CENH3.2 of cowpea is likely at an early stage of pseudogenization and less likely undergoing subfunctionalization.

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Most Hieracium subgenus Pilosella species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic Hieracium by targeting mutations in the endogenous PHYTOENE DESATURASE (PDS) gene using Agrobacterium-mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of PDS knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify PDS gene edits. Up to 86% of seeds produced albino seedlings with complete PDS knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing Cas9 segregation from the PDS target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic Hieracium, enabled development of the discussed strategies for future identification of causal apomixis genes.

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BACKGROUND: The legume cowpea (Vigna unguiculata L.) is extensively grown in sub-Saharan Africa. Cowpea, like many legumes has proved recalcitrant to plant transformation. A rapid transient leaf assay was developed for testing gene expression and editing constructs prior to stable cowpea transformation, to accelerate cowpea and legume crop improvement. RESULTS: Attempts to develop a transient protoplast system for cowpea were unsuccessful. Leaflets from plants 3-4 weeks post-germination were age selected to establish a rapid Agrobacterium (Agro) infiltration-mediated transient system for efficacy testing of gene expression and CRISPR/Cas9 gene editing constructs. In planta, Agro-infiltration of leaflets with fluorescent expression constructs, resulted in necrosis. By contrast, Agro-infiltration of detached leaflets with an Arabidopsis (At) ubiquitin3 promoter:ZsGreen construct, followed by culture on solid nutrient medium resulted in fluorescence in over 48% of leaf cells. Expression efficiency was leaf age-dependent. Three cowpea meiosis genes were identified for CRISPR/Cas9 gene-editing, with the forward aim of meiosis-knock out for asexual seed induction in cowpea. Constructs were designed and tested containing candidate gene-specific guide RNAs, expressed using either the cowpea or Arabidopsis U6 promoters with Cas9 expression directed by either the Arabidopsis 40S ribosomal protein or parsley ubiquitin4-2 promoters. Leaflets were infiltrated with test gene-editing constructs and analytical methods developed to identify gene-specific mutations. A construct that produced mutations predicted to induce functional knockout of in the VuSPO11-1 meiosis gene was tested for efficacy in primary transgenic cowpea plants using a previously established stable transformation protocol. Vuspo11-1 mutants were identified, that cytologically phenocopied spo11-1 mutants previously characterized in Arabidopsis, and rice. Importantly, a biallelic male and female sterile mutant was identified in primary transgenics, exhibiting the expected defects in 100% of examined male and female meiocytes. CONCLUSION: The transient, detached cowpea leaf assay, and supporting analytical methods developed, provide a rapid and reproducible means for testing gene expression constructs, and constructs for inducing mutagenesis in genes involved in both vegetative and reproductive developmental programs. The method and tested editing constructs and components have potential application for a range of crop legumes.

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Apomixis in Hieracium praealtum follows a developmental pathway of apospory, where an unreduced embryo sac develops from a somatic ovule cell without meiosis. The avoidance of meiosis together with fertilization-independent seed formation leads to clonal progeny genetically identical to the maternal plant. We have previously described the initial developmental steps of aposporous embryo sac formation in H. praealtum and here, we cytologically observed more than 500 ovules with a focus on the later stages of embryo sac maturation. Aposporous embryo sac maturation is a stochastic process in H. praealtum with single or multiple embryo sacs formed, in addition to off-types and embryo sac abortion. The frequency of twin embryo sacs growing at the same rate is a rare event and, in most ovules, the additional embryo sac undergoes developmental arrest suggesting dominance or growth promotion of a single embryo sac. Observed deviations from the Polygonum-type embryo sac in H. praealtum indicate developmental plasticity during embryo sac maturation. Nevertheless, fertilization-independent seed formation successfully occurs.

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Apomixis results in asexual seed formation where progeny are identical to the maternal plant. In ovules of apomictic species of the Hieracium subgenus Pilosella, meiosis of the megaspore mother cell generates four megaspores. Aposporous initial (AI) cells form during meiosis in most ovules. The sexual pathway terminates during functional megaspore (FM) differentiation, when an enlarged AI undergoes mitosis to form an aposporous female gametophyte. Then, the mitotically programmed FM dies along with the three other megaspores by unknown mechanisms. Transcriptomes of laser-dissected AIs, ovule cells, and ovaries from apomicts and AI-deficient mutants were analyzed to understand the pathways involved. The steps leading to AI mitosis and sexual pathway termination were determined using antibodies against arabinogalactan protein epitopes found to mark both sexual and aposporous female gametophyte lineages at inception. At most, four AIs differentiated near developing megaspores. The first expanding AI cell to contact the FM formed a functional AI that underwent mitosis soon after megaspore degeneration. Transcriptome analyses indicated that the enlarged, laser-captured AIs were arrested in the S/G2 phase of the cell cycle and were metabolically active. Further comparisons with AI-deficient mutants showed that AIs were enriched in transcripts encoding homologs of genes involved in, and potentially antagonistic to, known FM specification pathways. We propose that AI and FM cell contact provides cues required for AI mitosis and megaspore degeneration. Specific candidates to further interrogate AI-FM interactions were identified here and include Hieracium arabinogalactan protein family genes.

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Posttranscriptional gene silencing (PTGS) of transgenes involves abundant 21-nucleotide small interfering RNAs (siRNAs) and low-abundance 22-nucleotide siRNAs produced from double-stranded RNA (dsRNA) by DCL4 and DCL2, respectively. However, DCL2 facilitates the recruitment of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) to ARGONAUTE 1-derived cleavage products, resulting in more efficient amplification of secondary and transitive dsRNA and siRNAs. Here, we describe a reporter system where RDR6-dependent PTGS is initiated by restricted expression of an inverted-repeat dsRNA specifically in the Arabidopsis (Arabidopsis thaliana) root tip, allowing a genetic screen to identify mutants impaired in RDR6-dependent systemic PTGS. Our screen identified dcl2 but not dcl4 mutants. Moreover, grafting experiments showed that DCL2, but not DCL4, is required in both the source rootstock and the recipient shoot tissue for efficient RDR6-dependent systemic PTGS. Furthermore, dcl4 rootstocks produced more DCL2-dependent 22-nucleotide siRNAs than the wild type and showed enhanced systemic movement of PTGS to grafted shoots. Thus, along with its role in recruiting RDR6 for further amplification of PTGS, DCL2 is crucial for RDR6-dependent systemic PTGS.

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Background and Aims: Apomixis, or asexual seed formation, in polyploid Hieracium subgenus Pilosella species results in clonal progeny with a maternal genotype. An aposporous embryo sac forms mitotically from a somatic cell, without prior meiosis, while embryo and endosperm formation is fertilization independent (autonomous). The latter two developmental components are tightly linked in Hieracium . Recently, two plants, AutE196 and AutE24, were identified from two different crosses. Both form embryo sacs via the sexual route by undergoing meiosis, and embryo development requires fertilization; however, 18 % of embryo sacs can undergo autonomous endosperm (AutE) formation. This study investigated the qualitative and quantitative inheritance of the AutE trait and factors influencing phenotype expressivity. An additional focus was to identify the linkage group bearing the AutE locus in AutE196. Methods: Crosses and cytology were used to examine the inheritance of AutE from AutE24 and AutE196, and to reintroduce apomictic components into AutE plants, thereby changing the ploidy of developing embryo sacs and increasing the dosage of AutE loci. Markers from a Hieracium apomict linkage map were examined within a backcrossed AutE196 mapping population to identify the linkage group containing the AutE196 locus. Key Results: Qualitative autonomous endosperm in the AutE24 line was conferred by a single dominant locus, and the trait was transmitted through male and female gametes in AutE196 and AutE24. Expressivity of the trait did not significantly increase when AutE loci from AutE196 and AutE24 were both present in the progeny, within embryo sacs formed via apospory, or sexually derived embryo sacs with increased ploidy. It remains unclear if these are identical loci. Conclusions: The qualitative trait of autonomous endosperm formation is conferred by single dominant loci in AutE196 and AutE24. High expressivity of autonomous endosperm formation observed in apomicts requires additional genetic factors. Potential candidates may be signals arising from fertilization-independent embryo formation.

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BACKGROUND: Application of apomixis, or asexual seed formation, in crop breeding would allow rapid fixation of complex traits, economizing improved crop delivery. Identification of apomixis genes is confounded by the polyploid nature, high genome complexity and lack of genomic sequence integration with reproductive tissue transcriptomes in most apomicts. RESULTS: A genomic and transcriptomic resource was developed for Hieracium subgenus Pilosella (Asteraceae) which incorporates characterized sexual, apomictic and mutant apomict plants exhibiting reversion to sexual reproduction. Apomicts develop additional female gametogenic cells that suppress the sexual pathway in ovules. Disrupting small RNA pathways in sexual Arabidopsis also induces extra female gametogenic cells; therefore, the resource was used to examine if changes in small RNA pathways correlate with apomixis initiation. An initial characterization of small RNA pathway genes within Hieracium was undertaken, and ovary-expressed ARGONAUTE genes were identified and cloned. Comparisons of whole ovary transcriptomes from mutant apomicts, relative to the parental apomict, revealed that differentially expressed genes were enriched for processes involved in small RNA biogenesis and chromatin silencing. Small RNA profiles within mutant ovaries did not reveal large-scale alterations in composition or length distributions; however, a small number of differentially expressed, putative small RNA targets were identified. CONCLUSIONS: The established Hieracium resource represents a substantial contribution towards the investigation of early sexual and apomictic female gamete development, and the generation of new candidate genes and markers. Observed changes in small RNA targets and biogenesis pathways within sexual and apomictic ovaries will underlie future functional research into apomixis initiation in Hieracium.

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KEY MESSAGE: Cowpea reproductive tools. Vigna unguiculata L. Walp. (cowpea) is recognized as a major legume food crop in Africa, but seed yields remain low in most varieties adapted to local conditions. The development of hybrid cowpea seed that could be saved after each generation, enabling significant yield increases, will require manipulation of reproductive development from a sexual to an asexual mode. To develop new technologies that could support the biotechnological manipulation of reproductive development in cowpea, we examined gametogenesis and seed formation in two transformable, African-adapted, day-length-insensitive varieties. Here, we show that these two varieties exhibit distinct morphological and phenological traits but share a common developmental sequence in terms of ovule formation and gametogenesis. We present a reproductive calendar that allows prediction of male and female gametogenesis on the basis of sporophytic parameters related to floral bud size and reproductive organ development, determining that gametogenesis occurs more rapidly in the anther than in the ovule. We also show that the mode of megagametogenesis is of the Polygonum-type and not Oenothera-type, as previously reported. Finally, we developed a whole-mount immunolocalization protocol and applied it to detect meiotic proteins in the cowpea megaspore mother cell, opening opportunities for comparing the dynamics of protein localization during male and female meiosis, as well as other reproductive events in this emerging legume model system.

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In plants, embryogenesis generally occurs through the sexual process of double fertilization, which involves a haploid sperm cell fusing with a haploid egg cell to ultimately give rise to a diploid embryo. Embryogenesis can also occur asexually in the absence of fertilization, both in vitro and in vivo. Somatic or gametic cells are able to differentiate into embryos in vitro following the application of plant growth regulators or stress treatments. Asexual embryogenesis also occurs naturally in some plant species in vivo, from either ovule cells as part of a process defined as apomixis, or from somatic leaf tissue in other species. In both in vitro and in vivo asexual embryogenesis, the embryo precursor cells must attain an embryogenic fate without the act of fertilization. This review compares the processes of in vitro and in vivo asexual embryogenesis including what is known regarding the genetic and epigenetic regulation of each process, and considers how the precursor cells are able to change fate and adopt an embryogenic pathway.

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Evidence is presented for the role of a mitochondrial ribosomal (mitoribosomal) L18 protein in cell division, differentiation, and seed development after the characterization of a recessive mutant, heart stopper (hes). The hes mutant produced uncellularized endosperm and embryos arrested at the late globular stage. The mutant embryos differentiated partially on rescue medium with some forming callus. HES (At1g08845) encodes a mitochondrially targeted member of a highly diverged L18 ribosomal protein family. The substitution of a conserved amino residue in the hes mutant potentially perturbs mitoribosomal function via altered binding of 5S rRNA and/or influences the stability of the 50S ribosomal subunit, affecting mRNA binding and translation. Consistent with this, marker genes for mitochondrial dysfunction were up-regulated in the mutant. The slow growth of the endosperm and embryo indicates a defect in cell cycle progression, which is evidenced by the down-regulation of cell cycle genes. The down-regulation of other genes such as EMBRYO DEFECTIVE genes links the mitochondria to the regulation of many aspects of seed development. HES expression is developmentally regulated, being preferentially expressed in tissues with active cell division and differentiation, including developing embryos and the root tips. The divergence of the L18 family, the tissue type restricted expression of HES, and the failure of other L18 members to complement the hes phenotype suggest that the L18 proteins are involved in modulating development. This is likely via heterogeneous mitoribosomes containing different L18 members, which may result in differential mitochondrial functions in response to different physiological situations during development.

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BACKGROUND AND AIMS: Apomixis in plants generates clonal progeny with a maternal genotype through asexual seed formation. Hieracium subgenus Pilosella (Asteraceae) contains polyploid, highly heterozygous apomictic and sexual species. Within apomictic Hieracium, dominant genetic loci independently regulate the qualitative developmental components of apomixis. In H. praealtum, LOSS OF APOMEIOSIS (LOA) enables formation of embryo sacs without meiosis and LOSS OF PARTHENOGENESIS (LOP) enables fertilization-independent seed formation. A locus required for fertilization-independent endosperm formation (AutE) has been identified in H. piloselloides. Additional quantitative loci appear to influence the penetrance of the qualitative loci, although the controlling genes remain unknown. This study aimed to develop the first genetic linkage maps for sexual and apomictic Hieracium species using simple sequence repeat (SSR) markers derived from expressed transcripts within the developing ovaries. METHODS: RNA from microdissected Hieracium ovule cell types and ovaries was sequenced and SSRs were identified. Two different F1 mapping populations were created to overcome difficulties associated with genome complexity and asexual reproduction. SSR markers were analysed within each mapping population to generate draft linkage maps for apomictic and sexual Hieracium species. KEY RESULTS: A collection of 14 684 Hieracium expressed SSR markers were developed and linkage maps were constructed for Hieracium species using a subset of the SSR markers. Both the LOA and LOP loci were successfully assigned to linkage groups; however, AutE could not be mapped using the current populations. Comparisons with lettuce (Lactuca sativa) revealed partial macrosynteny between the two Asteraceae species. CONCLUSIONS: A collection of SSR markers and draft linkage maps were developed for two apomictic and one sexual Hieracium species. These maps will support cloning of controlling genes at LOA and LOP loci in Hieracium and should also assist with identification of quantitative loci that affect the expressivity of apomixis. Future work will focus on mapping AutE using alternative populations.

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Arabidopsis END1-LIKE (AtEND1) was identified as a homolog of the barley endosperm-specific gene END1 and provides a model for the study of this class of genes and their products. The END1 is expressed in the endosperm transfer cells (ETC) of grasses. The ETC are responsible for transfer of nutrients from maternal tissues to the developing endosperm. Identification of several ETC-specific genes encoding lipid transfer proteins (LTP), including the END1, provided excellent markers for identification of ETC during seed development. To understand how AtEND1 forms complexes with lipid molecules, a three-dimensional (3D) molecular model was generated and reconciled with AtEND1 function. The spatial and temporal expression patterns of AtEND1 were examined in transgenic Arabidopsis plants transformed with an AtEND1 promoter-GUS fusion construct. The AtEND1 promoter was found to be seed and pollen specific. In contrast to ETC-specific expression of homologous genes in wheat and barley, expression of AtEND1 is less specific. It was observed in ovules and a few gametophytic tissues. A series of AtEND1 promoter deletions fused to coding sequence (CDS) of the uidA were transformed in Arabidopsis and the promoter region responsible for AtEND1 expression was identified. A 163 bp fragment of the promoter was found to be sufficient for both spatial and temporal patterns of expression reflecting that of AtEND1. Our data suggest that AtEND1 could be used as a marker gene for gametophytic tissues and developing endosperm. The role of the gene is unclear but it may be involved in fertilization and/or endosperm cellularization.

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Apomixis (asexual seed formation) is the result of a plant gaining the ability to bypass the most fundamental aspects of sexual reproduction: meiosis and fertilization. Without the need for male fertilization, the resulting seed germinates a plant that develops as a maternal clone. This dramatic shift in reproductive process has been documented in many flowering plant species, although no major seed crops have been shown to be capable of apomixis. The ability to generate maternal clones and therefore rapidly fix desirable genotypes in crop species could accelerate agricultural breeding strategies. The potential of apomixis as a next-generation breeding technology has contributed to increasing interest in the mechanisms controlling apomixis. In this review, we discuss the progress made toward understanding the genetic and molecular control of apomixis. Research is currently focused on two fronts. One aims to identify and characterize genes causing apomixis in apomictic species that have been developed as model species. The other aims to engineer or switch the sexual seed formation pathway in non-apomictic species, to one that mimics apomixis. Here we describe the major apomictic mechanisms and update knowledge concerning the loci that control them, in addition to presenting candidate genes that may be used as tools for switching the sexual pathway to an apomictic mode of reproduction in crops.

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Over 200 imprinted genes in rice endosperm are known, but the mechanisms modulating their parental allele-specific expression are poorly understood. Here we use three imprinted genes, OsYUCCA11, yellow2-like and ubiquitin hydrolase, to show that differential DNA methylation and tri-methylation of histone H3 lysine 27 (H3K27me3 ) in the promoter and/or gene body influences allele-specific expression or the site of transcript initiation. Paternal expression of OsYUCCA11 required DNA methylation in the gene body whereas the gene body of the silenced maternal allele was hypomethylated and marked with H3K27me3 . These differential markings mirror those proposed to modulate paternal expression of two Arabidopsis genes, PHERES1 and a YUCCA homolog, indicating conservation of imprinting mechanisms. At yellow2-like, DNA hypomethylation in the upstream flanking region resulted in maternal transcripts that were longer than paternal transcripts; the maternal transcript initiation site was marked by DNA methylation in the paternal allele, and transcription initiated ~700 bp downstream. The paternal allele of an ubiquitin hydrolase gene exhibited gene body DNA methylation and produced full-length transcripts, while the maternal allele was hypomethylated in the 5' gene body and transcripts initiated from a downstream promoter. Inhibition of DNA methylation by 5-azacytidine or zebularine activated the long transcripts from yellow2-like and enhanced expression of the short transcripts from the ubiquitin hydrolase in seedlings, indicating that DNA methylation prevents transcript initiation from cryptic promoters. These observations suggest a paradigm whereby maternal genome hypomethylation is associated with the production of distinct transcripts, potentially diversifying the gene products from the two alleles.

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The formation of female gametes in plants occurs within the ovule, a floral organ that is also the precursor of the seed. Unlike animals, plants lack a typical germline separated from the soma early in development and rely on positional signals, including phytohormones, mobile mRNAs and sRNAs, to direct diploid somatic precursor cells onto a reproductive program. In addition, signals moving between plant cells must overcome the architectural limitations of a cell wall which surrounds the plasma membrane. Recent studies have addressed the molecular and histological signatures of young ovule cells and indicate that dynamic cell wall changes occur over a short developmental window. These changes in cell wall properties impact signal flow and ovule cell identity, thereby aiding the establishment of boundaries between reproductive and somatic ovule domains.

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Apomixis or asexual seed formation in Hieracium praealtum (Asteraceae) is controlled by two independent dominant loci. One of these, the LOSS OF APOMEIOSIS (LOA) locus, controls apomixis initiation, mitotic embryo sac formation (apospory) and suppression of the sexual pathway. The LOA locus is found near the end of a hemizygous chromosome surrounded by extensive repeats extending along the chromosome arm. Similar apomixis-carrying chromosome structures have been found in some apomictic grasses, suggesting that the extensive repetitive sequences may be functionally relevant to apomixis. Fluorescence in situ hybridization (FISH) was used to examine chromosomes of apomeiosis deletion mutants and rare recombinants in the critical LOA region arising from a cross between sexual Hieracium pilosella and apomictic H. praealtum. The combined analyses of aposporous and nonaposporous recombinant progeny and chromosomal karyotypes were used to determine that the functional LOA locus can be genetically separated from the very extensive repeat regions found on the LOA-carrying chromosome. The large-scale repetitive sequences associated with the LOA locus in H. praealtum are not essential for apospory or suppression of sexual megasporogenesis (female meiosis).

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Hieracium praealtum forms seeds asexually by apomixis. During ovule development, sexual reproduction initiates with megaspore mother cell entry into meiosis and formation of a tetrad of haploid megaspores. The sexual pathway ceases when a diploid aposporous initial (AI) cell differentiates, enlarges, and undergoes mitosis, forming an aposporous embryo sac that displaces sexual structures. Embryo and endosperm development in aposporous embryo sacs is fertilization independent. Transcriptional data relating to apomixis initiation in Hieracium spp. ovules is scarce and the functional identity of the AI cell relative to other ovule cell types is unclear. Enlarging AI cells with undivided nuclei, early aposporous embryo sacs containing two to four nuclei, and random groups of sporophytic ovule cells not undergoing these events were collected by laser capture microdissection. Isolated amplified messenger RNA samples were sequenced using the 454 pyrosequencing platform and comparatively analyzed to establish indicative roles of the captured cell types. Transcriptome and protein motif analyses showed that approximately one-half of the assembled contigs identified homologous sequences in Arabidopsis (Arabidopsis thaliana), of which the vast majority were expressed during early Arabidopsis ovule development. The sporophytic ovule cells were enriched in signaling functions. Gene expression indicative of meiosis was notably absent in enlarging AI cells, consistent with subsequent aposporous embryo sac formation without meiosis. The AI cell transcriptome was most similar to the early aposporous embryo sac transcriptome when comparing known functional annotations and both shared expressed genes involved in gametophyte development, suggesting that the enlarging AI cell is already transitioning to an embryo sac program prior to mitotic division.

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